Prodrugs of excitatory amino acids

ABSTRACT

This invention relates to synthetic excitatory amino acid prodrugs and processes for their preparation. The invention further relates to methods of using, and pharmaceutical compositions comprising, the compounds for the treatment of neurological disorders and psychiatric disorders.

This application is a continuation of U.S. patent application Ser. No.10/250,448, filed Nov. 12, 2003 now U.S. Pat. No. 7,256,217.

This invention relates to synthetic excitatory amino acid prodrugs (andtheir pharmaceutically acceptable salts) and processes for theirpreparation. The invention further relates to methods of using, andpharmaceutical compositions comprising, the compounds for the treatmentof neurological disorders and psychiatric disorders.

Treatment of neurological or psychiatric disorders, such as anxietydisorder, have been linked to selective activation of metabotropicexcitatory amino acid receptors such as (+)-2-aminobicyclo [3.1.0]hexane-2,6-dicarboxylic acid, also known as LY354740 which is disclosedin U.S. Pat. No. 5,750,566 (the '566 patent) issued May 12, 1998 is anactive MGLUR2 receptor agonist, CNS Drug Reviews, 5 pgs. 1-12 (1999).

The present invention provides for a prodrug form of LY354740 whichenhances the in vivo potency of LY354740, producing higher oral exposureof the parent compound. In addition, when compounds of the presentinvention are administered, no circulating level of prodrug was detectedwith high in vitro bioconversion to the parent molecule. Further, thepeptide prodrugs are stable under all ranges of pH and are nontoxic.Compounds of the present invention represent the best approach formaintaining LY354740-like safety and efficacy in humans with increasedoral bioavailability. Preclinical studies with,(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride, the compound of thepresent invention, has shown greatly enhanced oral potency in thetreatment of anxiety without the attendant problems of toxicity,instability at desired pH ranges and low in vivo conversion.

Accordingly, the present invention provides a compound of formula I

wherein

R¹³, R¹⁴ and R¹⁷ are hydrogen;

or a pharmaceutically acceptable salt thereof.

Compounds of the invention have been found to be useful prodrugs forLY354740 a selective agonist of metabotropic glutamate receptors and aretherefore useful in the pharmaceutical treatment of diseases of thecentral nervous system such as neurological diseases, for exampleneurodegenerative diseases, and as antipsychotic, anxiolytic,drug-withdrawal, antidepressant, anticonvulsant, analgesic andanti-emetic agents.

It will be appreciated that the compounds of formula (I) contain atleast four asymmetric carbon atoms, three being in the cyclopropane ringand one being at the α-carbon of the amino acid group. Accordingly, thecompounds of the invention may exist in and be isolated inenantiomerically pure form, in racemic form, or in a diastereoisomericmixture.

The amino acid moiety preferably has the natural amino acidconfiguration, i.e. the L-configuration relative to D-glycerol aldehyde.

The present invention includes pharmaceutically acceptable salts of thecompound of formula I. These salts can exist in conjunction with theacidic or basic portion of the molecule and can exist as acid addition,primary, secondary, tertiary, or quaternary ammonium, alkali metal, oralkaline earth metal salts. Generally, the acid addition salts areprepared by the reaction of an acid with a compound of formula I. Thealkali metal and alkaline earth metal salts are generally prepared bythe reaction of the hydroxide form of the desired metal salt with acompound of formula I.

Some particular salts provide certain formulation advantages due totheir crystalline form. Non-crystalline forms of compounds may beamorphous and hygroscopic. Crystalline forms of pharmaceutical compoundsare sometimes more desirable because they are not amorphous.

A particular pharmaceutically acceptable salt of the peptide of formulaI is (1S,2S,5R,6S)-2- [(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride salt.

Another particular pharmaceutically acceptable salt of the peptide offormula I is (1S,2S,5R,6S)-2- [(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid methane sulfonate salt.

Acids commonly employed to form such salts include inorganic acids, forexample hydrochloric, hydrobromic, nitric, sulphuric or phosphoricacids, or with organic acids, such as organic carboxylic acids, forexample, glycollic, maleic, hydroxymaleic, fumaric, malic, tartaric,citric, salicyclic, o-acetoxybenzoic, or organic sulphonic,2-hydroxyethane sulphonic, toluene-p-sulphonic, methane-sulfonic ornaphthalene-2-sulphonic acid.

In addition to pharmaceutically-acceptable salts, other salts areincluded in the invention. They may serve as intermediates in thepurification of compounds or in the preparation of other, for examplepharmaceutically-acceptable, acid addition salts, or are useful foridentification, characterization or purification.

A variety of physiological functions have been shown to be subject toinfluence by excessive or inappropriate stimulation of excitatory aminoacid transmission. The formula I compounds of the present invention arebelieved to have the ability to treat a variety of neurologicaldisorders in mammals associated with this condition, including acuteneurological disorder such as cerebral deficits subsequent to cardiacbypass surgery and grafting, stroke, cerebral ischemia, spinal cordtrauma, head trauma, perinatal hypoxia, cardiac arrest, and hypoglycemicneuronal damage. The formula I compounds are believed to have theability to treat a variety of chronic neurological disorders, such asAlzheimer's disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, ocular damage and retinopathy, cognitivedisorders, and idiopathic and drug-induced Parkinson's. The presentinvention also provides methods for treating these disorders whichcomprises administering to a patient in need thereof an effective amountof a compound of formula I or a pharmaceutically acceptable saltthereof.

The formula I compounds of the present invention treat a variety ofother neurological disorders in patients that are associated withglutamate dysfunction, including muscular spasms, convulsions, migraineheadaches, urinary incontinence, pain, premenstrual dysphoric disorder(PDD), psychosis, (such as schizophrenia), drug tolerance and withdrawal(such as nicotine, opiates and benzodiazepines), anxiety and relateddisorders, emesis, brain edema, chronic pain, and tardive dyskinesia.The formula I compounds are also useful as antidepressant and analgesicagents. Therefore, the present invention also provides methods fortreating these disorders which comprise administering to a patient inneed thereof an effective amount of the compound of formula I, or apharmaceutically acceptable salt thereof.

A compound of Formula I may be made by a process which is analogous toone known in the chemical art for the production of structurallyanalogous heterocyclic compounds or by a novel process described herein.Such processes and intermediates useful for the manufacture of acompound of Formula I as defined above are provided as further featuresof the invention and are illustrated by the following procedures inwhich, unless otherwise specified, the meanings of the generic radicalsare as defined above.

(A) For a compound of formula I in which R¹³, R¹⁴, and R¹⁷ are hydrogen(a di-acid), deprotecting the amine group of a compound of formula I

where R¹⁷ is tert-butoxy carbonyl or a nitrogen protecting group, withan acid as described in the General Procedures for Examples 3 and 4.

(B) For a compound of formula I in which R¹³ and R¹⁴ are both hydrogen(a di-acid), deprotecting a compound of formula I where R¹³ and R¹⁴ arenot both hydrogen as described in Scheme 2.

(C) For a compound of formula I in which R¹³ and R¹⁴ are not bothhydrogen, amidating a compound of formula II

with a corresponding amino acid of formula III.HOOCCHCH₃NHR¹⁷  IIIin which p is O or any integer from 1-10 and R¹⁷ is tert-butoxy carbonylor a nitrogen-protecting group as described in the General Procedure forExample 1.

(D) For a compound of formula II where R¹³ and R¹⁴ are not hydrogen,where R¹³ and R¹⁴ may be a carboxy-protecting ester group (a di-ester),esterifying a compound of formula II where R¹³ and R¹⁴ are both hydrogen(a di-acid).

(E) For a compound of formula II in which R¹³ and R¹⁴ are not hydrogen(a di-ester), deprotecting a compound of formula IV

where R^(m) is a nitrogen protecting group, as described in Preparation2.

(F) For a compound of formula II where R¹³ and R¹⁴ are not both hydrogen(a di-ester), esterifying a compound of formula IV, as described inPreparation 2.

(G) For a compound of formula IV where R¹³ and R¹⁴ are both hydrogen (adi-acid), protecting the amine group of a compound of formula II asdescribed in Preparation 1.

The term “nitrogen protecting group,” as used herein, refers to thosegroups intended to protect or block the nitrogen group againstundesirable reactions during synthetic procedures. Choice of thesuitable nitrogen protecting group used will depend upon the conditionsthat will be employed in subsequent reaction steps wherein protection isrequired, as is well within the knowledge of one of ordinary skill inthe art. Commonly used nitrogen protecting groups are disclosed in T. W.Greene and P. G. M. Wuts, Protective Groups In Organic Synthesis, 2^(nd)Ed. (John Wiley & Sons, New York (1991)).

The term “carboxy-protecting group” as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups of the compound. Particular valuesinclude, for example, methyl, ethyl, tert-butyl, benzyl, methoxymethyl,trimethylsilyl, and the like. Further examples of such groups may befound in T. W. Greene and P. G. M. Wuts, Protecting Groups in OrganicSynthesis, 3rd. Ed. (John Wiley & Sons, N.Y. (1999)). The ester isdecomposed by using a conventional procedure which does not affectanother portion of the molecule.

Whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of Formula I is required, it is obtainedby reacting the acid of Formula I with a physiologically acceptable baseor by reacting a basic compound of Formula I with a physiologicallyacceptable acid or by any other conventional procedure.

The term “C₁-C₁₀ alkyl” represents a straight, branched, or cyclic alkylchain having from one to ten carbon atoms.

The term “C₂-C₁₀ alkenyl” represents straight or branched unsaturatedalkyl chains having from two to ten carbon atoms, and having one or morecarbon-carbon double bond, such as, dienes and trienes. This group alsoincludes both E and Z isomers.

The term “aryl” represents groups such as phenyl, substituted phenyl,and naphthyl. The term “arylalkyl” represents a C₁-C₄ alkyl groupbearing one or more aryl groups.

The term “affecting” refers to a formula I compound acting as an agonistat an excitatory amino acid receptor. The term “excitatory amino acidreceptor” refers to a metabotropic glutamate receptor, a receptor thatis coupled to cellular effectors via GTP-binding proteins. The term“cAMP-linked metabotropic glutamate receptor” refers to a metabotropicreceptor that is coupled to inhibition of adenylate cyclase activity.

The term “neurological disorder” refers to both acute and chronicneurodegenerative conditions, including cerebral deficits subsequent tocardiac bypass surgery and grafting, cerebral ischemia (for examplestroke resulting from cardiac arrest), spinal cord trauma, head trauma,Alzheimer's Disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, perinatal hypoxia, hypoglycemic neuronal damage,ocular damage and retinopathy, cognitive disorders, idiopathic anddrug-induced Parkinson's Disease. This term also includes otherneurological conditions that are caused by glutamate dysfunction,including muscular spasms, migraine headaches, urinary incontinence,drug tolerance, withdrawal, and cessation (i.e. opiates,benzodiazepines, nicotine, cocaine, or ethanol), smoking cessation,emesis, brain edema, chronic pain, sleep disorders, convulsions,Tourette's syndrome, attention deficit disorder, and tardive dyskinesia.

The term “psychiatric disorder,” refers to both acute and chronicpsychiatric conditions, including schizophrenia, anxiety and relateddisorders (e.g. panic attack and stress-related cardiovasculardisorders), depression, bipolar disorders, psychosis, and obsessivecompulsive disorders.

A particular aspect of the present invention includes a method foraffecting the cAMP-linked metabotropic glutamate receptors in a patient,which comprises administering to a patient requiring modulatedexcitatory amino acid neurotransmission a pharmaceutically-effectiveamount of a compound of formula I.

Another particular aspect of the present invention includes a method ofadministering an effective amount of a compound of formula II, where R¹³and R¹⁴ are both hydrogen (a di-acid), which comprises administering toa patient requiring modulated excitatory amino acid neurotransmission apharmaceutically effective amount of a compound of formula I.

Another particular aspect of the present invention includes a method fortreating a psychiatric disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of formula I.

Another particular aspect of the present invention includes a method fortreating a neurological disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of formula I.

A preferred method for treating a psychiatric disorder in a patientcomprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid psychiatric disorder is schizophrenia, anxiety and relateddisorders, depression, dipolar disorders, psychosis, and obsessivecompulsive disorders.

A preferred method for treating a neurological disorder in a patientcomprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid neurological disorder is cerebral deficits subsequent to cardiacbypass and grafting; cerebral ischemia; spinal cord trauma; head trauma;Alzheimer's Disease; Huntington's Chorea; amyotrophic lateral sclerosis;AIDS-induced dementia; perinatal hypoxia; hypoglycemic neuronal damage;ocular damage and retinopathy; cognitive disorders; idiopathic anddrug-induced Parkinsons' Disease; muscular spasms; migraine headaches;urinary incontinence; drug tolerance, withdrawal, and cessation; smokingcessation; emesis; brain edema; chronic pain; sleep disorders;convulsions; Tourette's syndrome; attention deficit disorder; andtardive dyskinesia.

A more preferred method for treating a psychiatric disorder in a patientcomprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid psychiatric disorder is anxiety and related disorders.

A more preferred method for treating a neurological disorder in apatient comprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid neurological disorder is drug tolerance, withdrawal, and cessation;or smoking cessation.

An additional aspect of the present invention is a compound of formulaI, or a pharmaceutically acceptable salt thereof, for use as apharmaceutical.

Another aspect of the present invention includes the use of a compoundof formula I, or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament for treating neurological or psychiatricdisorders.

As used herein the term “effective amount” refers to the amount or doseof the compound, upon single or multiple dose administration to thepatient, which provides the desired effect in the patient underdiagnosis or treatment.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances. For example, a typicaldaily dose may contain from about 25 mg to about 300 mg of the activeingredient. The compounds can be administered by a variety of routesincluding oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, bucal or intranasal routes. Alternatively, the compoundmay be administered by continuous infusion.

As used herein the term “patient” refers to a mammal, such as a mouse,guinea pig, rat, dog or human. It is understood that the preferredpatient is a human.

The term “treating” (or “treat”) as used herein includes its generallyaccepted meaning which encompasses prohibiting, preventing, restraining,and slowing, stopping, or reversing progression of a resultant symptom.As such, the methods of this invention encompass both therapeutic andprophylactic administration.

If not commercially available, the necessary starting materials for theabove procedures may be made by procedures which are selected fromstandard techniques of organic and heterocyclic chemistry, techniqueswhich analogous to the syntheses of known, structurally similarcompounds, and the procedures described in the Examples, including novelprocedures.

A further aspect of the present invention provides for a method ofadministering an effective amount of a compound of formula II, where R¹³and R¹⁴ are both hydrogen (a di-acid), which comprises administering toa patient requiring modulated excitatory amino acid neurotransmission apharmaceutically-effective amount of a compound of formula I.

Compounds of formula I are converted via enzymatic or hydrolytic processin vivo, to form compounds of formula II, where R¹³ and R¹⁴ are bothhydrogen (a di-acid), as shown in Scheme 1 below.

In particular, a crystalline form of a compound of formula I may beprepared according to the route outlined in Scheme 2 below in which eachof R¹³ and R¹⁴, respectively, represents a value defined for the groupsR¹³ and R¹⁴. The process described in Scheme 2 is a synthesis method forthe preparation of a crystalline hydrochloride salt form of a compoundof formula I and a methanesulfonate salt form of a compound of formulaI.

In scheme 2 above, the monohydrate of II, where R¹³ and R¹⁴ are bothhydrogen (a di-acid), is treated with thionyl chloride and methanolaffording the corresponding di-ester of II. Alternatively, catalytichydrochloric acid may be used in place of thionylchloride. The di-ester,formula II, is amidated with a compound of formula III usingdicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCl) or isobutylchloroformate as a coupling agent to afford a di-ester protectedpeptidyl compound of formula I. This transformation could also beachieved using the acid chloride or by using a variety of other peptidecoupling reagents, for example, diphenyl chlorophosphate and2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),bis(2-oxo-3-oxazolidinyl)phosphinic chloride andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.

The hydrolysis of the di-ester protected peptidyl compound of formula Iwith a suitable base such as lithium hydroxide or sodium hydroxide inTHF affords the di-acid protected peptidyl compound of formula I, whereR¹³ and R¹⁴ are both hydrogen (a di-acid). The di-acid protectedpeptidyl compound of formula I may be deprotected with a mineral ororganic acid in a suitable solvent. Such conditions may produce thecorresponding acid salt of the di-acid peptidyl compound of formula I asan amorphous solid or, directly, a crystalline solid. In the case of anamorphous solid, subsequent crystallization may occur from suitablesolvents. For example, a di-acid protected peptidyl compound of formulaI when treated hydrogen chloride gas in ethyl acetate provides thedeprotected hydrochloride salt as an amorphous solid. The amorphoushydrochloride compound may then be crystallized from acetone and waterto afford the crystalline hydrochloride salt compound of formula I. Inthe case of a crystalline solid which is formed directly, filtration ofthe reaction mixture may afford the crystalline salt. The zwitterioniccompound of formula I is afforded by treatment of the crystallinehydrochloride salt of formula I with sodium hydroxide. It will beappreciated by one of ordinary skill in the art that a compound offormula I may be prepared in one procedure where the indicatedintermediates are not isolated.

The ability of compounds to modulate metabotropic glutamate receptorfunction may be demonstrated by examining their ability to influenceeither cAMP production (mGluR 2, 3, 4, 6, 7 or 8) or phosphoinositidehydrolysis (mGluR 1 or 5) in cells expressing these individual humanmetabotropic glutamate receptor (mGluR) subtypes. (D. D. Schoepp, etal., Neuropharmacol., 1996, 35, 1661-1672 and 1997, 36, 1-11).

The ability of formula I compounds to treat anxiety or a relateddisorder may be demonstrated using the well known fear potentiatedstartle and elevated plus maze models of anxiety described respectivelyin Davis, Psychopharmacology, 62:1; 1979 and Lister, Psychopharmacol,92:180-185; 1987

In Vitro Receptor Binding

To study the ability to affect receptor binding of compounds of thepresent invention in comparison to LY354740 displacement of a highaffinity mGluR2 antagonist radioligand [³H]LY341495 to cell membranesfrom human mGluR2 human mGluR3 and native rat brain tissues wasdetermined. (See, Ornstein P. L., Arnold M. B., Bleisch T. J., Wright R.A., Wheeler W. J., and Schoepp D. D., [³H]LY341495 a highly potent,selective and novel radioligand for labeling group II metabotropicreceptors. Bioorg. Med. Chem. Lett. 8: 1919-1922 (1998); and Johnson B.G., Wright R. A., Arnold M. B., Wheeler W. J., Ornstein P. L., andSchoepp D. D., [³H]LY341495 as a novel rapid filtration antagonistradioligand for group II metabotropic receptors: Characterization ofbinding to membranes of mGlu receptor subtype expressing cells.Neuropharmacology 38: 1519-1529 (1999))

As shown in Table 1 below, LY354740 displaced [³H]LY341495 binding torat forebrain membranes with a potency similar to that observed in humanrecombinant receptors. In contrast, the compound of formula I did notappreciably displace [³H]LY341495 binding to rat forebrain membranes atup to 10,000 mM.

TABLE 1 Comparison of receptor binding of compounds of the presentinvention with LY354740 Displacement of ³H-LY341495 binding Receptor(Ki, nM)(mean ± S.E., N = 3) Preparation LY354740 Formula I Human mGluR2 84.7 ± 11.1 >10,000 Human mGluR3 125.6 ± 4.8  >10,000 Rat Forebrain80.0 ± 7.3 >10,000In Vivo Actions in Rat Fear Potentiated Startle Anxiety Model

To study the oral potencies of compounds of the present invention incomparison to LY354740 in an mGlu2/3 receptor linked therapeutic animalmodel, studies in the rat fear-potentiated startle assay were performed.This model was specifically chosen, as it is highly sensitive to mGlu2/3agonists such as LY354740 and compounds of the present invention. (See,Helton D. R., Tizzano J. P., Monn J. A., Schoepp D. D., and Kallman M.J., Anxiolytic and side-effect profile of LY354740: A potent, highselective, orally active agonist for group II metabotropic glutamatereceptors, J. Pharmacol. Exp. Ther. 284: 651-660 (1998)). To verify thatthe actions of a compound of formula I in this model were mGlu2/3receptor mediated, as has been shown previously for LY354740 (See,Tizzano, J. P., Griffey K. I., Ornstein P. L., Monn J. A., and SchoeppD. D., Actions of mGlu receptor agonists on fear-conditioning versusfear-expression in rats, Neuropharmacology, 38:A45 (#144) (1999)), theability of LY341495 (an mGlu2/3 receptor antagonist) (See, Kingston A.E., Ornstein P. L., Wright R. A., Johnson B. G., Mayne N. G., Burnett J.P., Belagaje R., Wu S., and Schoepp D. D., LY341495 is a nanomolarpotent and selective antagonist for group II metabotropic glutamatereceptors, Neuropharmacology, 37: 1-12(1998)) to block compound-mediatedsuppression of fear-potentiated startle was also determined. As apositive control in each experiment, diazepam (0.6 mg/kg i.p.) was used.All experiments were performed in fed rats.

In the fear potentiated startle model, animals are exposed to a neutralstimulus such as light (conditioned stimulus) with an aversive stimulussuch as a shock (unconditioned stimulus). Following conditioning, whenthe animals are presented with a loud acoustic stimulus, larger startleresponses are elicited when the startle stimulus is preceded by light.

Diazepam and buspirone hydrochloride, which are clinically provenanxiolytics, are effective at reducing the fear (increased startleresponse) associated with the presentation of light in the fearpotentiated startle model, and in reducing the fear of open spaces inthe elevated plus maze model.

Male Long Evans rats (180-400 g) or male NIH Swiss mice (18-35 g) wereobtained from Harlan Sprague-Dawley, Cumberland, Ind., USA andacclimated at least 3 days before testing. Animals were housed at 23±2°C. (relative humidity 30% to 70%) and given Purina Certified Rodent Chowand water ad libitum. The photoperiod was 12 hours of light and 12 hoursof dark, with dark onset at approximately 1800 hours.

Test compounds were dissolved in a vehicle of purified water andneutralized with 5 N NaOH to a pH of 7-8 when applicable. Diazepam(Sigma Chemical Company, St. Louis, Mo.) was suspended in purified waterby the dropwise addition of Tween 80. Control animals received therespective vehicle.

SL-LAB (San Diego Instruments, San Diego, Calif.) chambers were used forconditioning sessions and for the production and recording of startleresponses. A classical conditioning procedure was used to producepotentiation of startle responses. Briefly, on the first 2 days, ratswere placed into dark startle chambers in which shock grids wereinstalled. Following a 5-minute acclimation period, each rat received a1 mA electric shock (500 ms) preceded by a 5 second presentation oflight (15 watt) which remained on for the duration of the shock. Tenpresentations of the light and shock were given in each conditioningsession, rats were gavaged with a solution of test compound of water andstartle testing sessions were conducted. A block of 10 consecutivepresentations of acoustic startle stimuli (110 dB, non-light-paired)were presented at the beginning of the session in order to minimize theinfluences of the initial rapid phase of habituation to the stimulus.This was followed by 20 alternating trials of the noise alone or noisepreceded by the light. Excluding the initial trial block, startleresponse amplitudes for each trial type (noise−alone vs. light+noise)were averaged for each rat across the entire test session.

As shown in the first row of Table 2, below, when given orally to fedrats, compounds of the present invention were active in the ratfear-potentiated startle test at 300 times lower doses when compared toLY354740. If this in vivo animal model data directly predicts humananxiety responses, compounds of the present invention would produceanxiolytic effects in humans at 300 fold lower doses than the parentcompound. Furthermore, the ability to produce a longer duration at lowerdoses when compared to parent may allow for once-a day dosing, asopposed to twice a day dosing.

In Vivo Exposure as Measured by Rat Plasma Concentration

To study the in vivo exposure of LY354740 following oral dosing ofcompounds of the present invention in comparison to LY354740, studiesmeasuring the plasma concentrations of LY354740 in rats were performed.

Mature Fischer 344 male rats (190-270 gram) were obtained from HarlanSprague-Dawley, Cumberland, Ind., USA and acclimated in the studyhousing for 3 days. On day 4 test compounds were dissolved in bufferedwater (1 mg/ml=test compound/20 mM potassium dihydrogen phosphate, pH=2)and given orally as a single 5 mg/kg dose. Blood samples were collectedthrough orbital sinus or cardiac puncture (last time point) at 0.5 and 1hour or, alternatively, 1 and 3 hours. Plasma samples were stored at−20° C. in the presence of phenylmethylsulfonyl fluoride, a proteaseinhibitor, prior to analysis. Plasma samples and internal standardcompounds were pretreated by solid phase extraction (SAX support,methanol/water/dilute acetic acid). As shown in the second row of Table2, below, the plasma concentrations (ng/ml) of LY354740 for each testcompound were determined by LC/MS/MS and are presented as a sum of theconcentrations at the 0.5 and 1 hour or, alternatively, 1 and 3 hoursample time points.

TABLE 2 Comparison LY354740 and compounds of the present invention inthe rat fear-potentiated startle assay Parameter Compound MeasuredLY354740 formula I MED (1 hour 3.0 mg/kg p.o. 0.01 mg/kg p.o. pre-treatement) Rat Exposure 466 ng/ml 7114 ng/ml (ng/ml of LY354740following 5 mg/kg p.o.)

As shown above in Tables 1 and 2, in vitro studies show that thecompounds of the present invention had no appreciable affinity per sefor mGlu2/3 receptors. This indicates that the in vivo pharmacology ofthis compound in rats and humans would likely reflect the conversion ofthe prodrug to the parent molecule, LY354740, which then acts at mGlu2/3receptors to produce a therapeutic effect. Further, in fact, when givenorally to rats, the compounds of the current invention exhibit a 15 foldincrease in plasma concentration of LY354740 when compared to LY354740.This demonstrates compounds of the present invention are converted toLY354740 in vivo.

The compounds of the present invention are preferably formulated priorto administration. Therefore, another aspect of the present invention isa pharmaceutical formulation comprising a compound of formula I, or apharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier, diluent, or excipient. Thepharmaceutical formulations may be prepared by procedures well-known byone of ordinary skill in the art. In making the compositions of thepresent invention, the active ingredient will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier, and maybe in the form of a capsule, sachet, paper, or other container. When thecarrier serves as a diluent, it may be a solid, semi-solid, or liquidmaterial which acts as a vehicle, excipient, or medium for the activeingredient. The compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols, ointments containing, for example, up to10% by weight of active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum, acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc,magnesium stearate, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents, or flavoring agents.Compositions of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 mg to about 500 mg active ingredient,preferably about 25 mg to about 300 mg active ingredient. As used hereinthe term “active ingredient” refers to a compound included within thescope of formula I.

The term “unit dosage form” refers to a physically discrete unitsuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical carrier, diluent, or excipient.

The following Examples further illustrate the compounds of the presentinvention and the methods for their synthesis. The Examples are notintended to be limiting to the scope of the invention in any respect,and should not be so construed. All experiments were run under apositive pressure of dry nitrogen or argon. All solvents and reagentswere purchased from commercial sources and used as received, unlessotherwise indicated. Dry tetrahydrofuran (THF) was obtained bydistillation from sodium or sodium benzophenone ketyl prior to use.Proton nuclear magnetic resonance (¹H NMR) spectra were obtained on aBruker Avance II bay-500 at 500 MHz, a Bruker Avance I bay-200 at 200MHz or a Varian Inova at 500 MHz. Electrospray mass spectroscopy (ESI)was performed on a Agilent MSD/B instrument using acetonitrile/aqueousammonium acetate as the mobile phase. Free atom bombardment massspectroscopy (FABMS) was performed on a VG ZAB-2SE instrument. Fielddesorption mass spectroscopy (FDMS) was performed using either a VG 70SEor a Varian MAT 731 instrument. Optical rotations were measured with aPerkin-Elmer 241 polarimeter. Chromatographic separation on a WatersPrep 500 LC was generally carried out using a linear gradient of thesolvents indicated in the text. The reactions were generally monitoredfor completion using thin layer chromatography (TLC). Thin layerchromatography was performed using E. Merck Kieselgel 60 F₂₅₄ plates, 5cm×10 cm, 0.25 mm thickness. Spots were detected using a combination ofUV and chemical detection (plates dipped in a ceric ammonium molybdatesolution [75 g of ammonium molybdate and 4 g of cerium (IV) sulfate in500 mL of 10% aqueous sulfuric acid] and then heated on a hot plate).Flash chromatography was performed as described by Still, et al. Still,Kahn, and Mitra, J. Org. Chem., 43, 2923 (1978). Elemental analyses forcarbon, hydrogen, and nitrogen were determined on a Control EquipmentCorporation 440 Elemental Analyzer, or were performed by the UniversidadComplutense Analytical Centre (Facultad de Farmacia, Madrid, Spain).Melting points were determined in open glass capillaries on a Gallenkamphot air bath melting point apparatus or a Büchi melting point apparatus,and are uncorrected.

The abbreviations, symbols and terms used in the examples have thefollowing meanings.

-   -   Ac=acetyl    -   Anal.=elemental analysis    -   Bn or Bzl=benzyl    -   Bu=butyl    -   BOC=butoxycarbonyl    -   calcd=calculated    -   D₂O=deuterium oxide    -   DCC=dicyclohexylcarbodiimide    -   DIBAL-H=diisobutyl aluminum hydride    -   DMAP=dimethylaminopyridine    -   DMF=dimethylformamide    -   DMSO=dimethylsulfoxide    -   EDC=N-ethyl-N′N′-dimethylaminopropyl carbodiimide    -   Et=ethyl    -   EtOH=ethanol    -   FAB=Fast Atom Bombardment (Mass Spectrascopy)    -   FDMS=field desorption mass spectrum    -   HOAt=1-hydroxy-7-azabenzotriazole    -   HOBt=1-hydroxybenzotriazole    -   HPLC=High Performance Liquid Chromatography    -   HRMS=high resolution mass spectrum    -   i-PrOH=isopropanol    -   IR=Infrared Spectrum    -   L=liter    -   Me=methyl    -   MeOH=methanol    -   MPLC=Medium Pressure Liquid Chromatography    -   Mp=melting point    -   MTBE=t-butyl methyl ether    -   NBS=N-bromosuccinimide    -   NMR=Nuclear Magnetic Resonance    -   Ph=phenyl    -   p.o.=oral administration    -   i-Pr=isopropyl    -   Rochelle's Salt=potassium sodium tartrate    -   SM=starting material    -   TBS=tert-butyldimethylsilyl    -   TEA=triethylamine    -   Temp.=temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   t-BOC=tert-butoxycarbonyl

Preparation 1 Synthesis of(1S,2S,5R,6S)-2-tert-Butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

A 1 L flask was charged with(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (24.4 g, 0.12 mol, 1 equiv), dioxane (200 mL) anddi-tert-butyl dicarbonate (52.4 g, 0.24 mol, 2.0 equiv). The suspensionwas vigorously stirred while 1N sodium hydroxide (420 mL, 3.5 equiv) wasadded. The mixture was stirred for 2 days, then 2.0 more equiv ofdi-tert-butyl dicarbonate were added and the reaction stirred for 3additional days at room temperature. After 5 total days of reaction,water (400 mL) was added to dissolve the salts. The aqueous layer wasextracted with ethyl acetate (4×100 mL) and acidified to pH 2 with 6 Nhydrochloric acid. The acidic aqueous phase was extracted with ethylether (6×200 mL). The combined ether extracts were washed with water(250 mL) and brine (250 mL). After drying over sodium sulfate, solventswere evaporated under vacuum to afford a foamy white solid (26.4 g). 77%Yield; mp 100-101° C. [α]_(D) ²⁵=−41.1° (c=1.0, MeOH). ¹H NMR(Methanol-d₄) δ: 4.98 (brs, 1H), 2.44 (dd, 1H, J=6.2, 2.6 Hz), 2.19-1.92(m, 4H), 1.62 (t, 1H, J=2.8 Hz) 1.43 (s, 9H), 1.29 (m, 1H). ¹³C NMR(Methanol-d₄) δ: 175.6, 175.2, 158.2, 60.1, 34.6, 31.9, 28.4, 27.2,25.6, 20.6. MS (Electrospray): 285.12.

Preparation 2 Synthesis of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride

(1S,2S,5R,6S)-2-tert-Butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (20 g, 0.07 mol, 1.0 equiv) was dissolved in 210 ml of drydimethylformamide and potassium carbonate (21.3 g, 0.154 mol, 2.2 equiv)was added at 0° C. under nitrogen. After 15 minutes, methyl iodide (17.6ml, 0.28 mol, 4.0 equiv) was added. The reaction mixture was warmed upslowly and stirred at room temperature for 3h. Water (200 ml) was addedand the aqueous phase was extracted with ethyl ether (4×75 ml each). Thecombined organic phase was washed with cold water (4×50 ml), and theaqueous phase extracted again with ethyl ether (2×50 ml). After dryingthe organic phase over sodium sulfate and evaporating under vacuum, afoamy solid((1S,2S,5R,6S)-2-tert-butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid-2,6-dimethyl ester) was obtained (19.2 g, 87% yield).

This compound was diluted with 150 ml of a saturated solution ofhydrogen chloride gas in ethyl acetate and the mixture vigorouslystirred for 1 hour (a white precipitate appeared within 15 minutes). Thesolid was filtered, rinsed with ethyl ether and thoroughly dried underhigh vacuum.

73% Yield; mp 193-194° C. [α]_(D) ²⁵=+22.2° (c=1.0, MeOH). ¹H NMR (D₂O)δ: 3.86 (s, 3H), 3.67 (s, 3H), 2.31-2.04 (m, 6H), 1.57 (m, 1H). ¹³C NMR(Methanol-d₄) δ: 171.9, 170.2, 65.6, 52.8, 51.2, 32.4, 29.9, 28.5, 26.2,20.7.

Alternative Synthesis of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride

Thionyl chloride (807 mL, 11.1 mol) was added to methanol (9.5 L) over aperiod of 1 h while maintaining the temperature between 2-20° C. Thesolution was maintained for 30 min, then(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (1.61 kg, 7.92 mol) was added. The resulting solution washeated to 47° C. and maintained between 47-50° C. for 17 h.Approximately 7.3 L of methanol was then removed by vacuum distillation(47-50° C., 240-275 mm Hg). The remaining methanol was removed byazeotropic distillation with t-butyl methyl ether (MTBE) at atmosphericpressure [added MTBE (10 L), removed 8.5 L; added MTBE (10 L), removed8.5 L; added MTBE (8 L), removed 5.1 L]. During the course of thedistillations a white solid began to precipitate from the solution.After completion of the distillations, MTBE (2 L) was added to theresulting slurry, and the slurry was cooled to 22° C. The solid wasfiltered, rinsed with MTBE (2 L) and dried under vacuum to afford 1.94kg (98%) of the title compound as a white solid.

Analysis Calculated for C₁₀H₁₆NO₄Cl: C, 48.10; H, 6.46; N, 5.61; Cl,14.20. Found: C, 47.88; H, 6.25; N, 5.57; Cl, 14.52.

General Procedure for the coupling reaction of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride with N-BOC-(L)-aminoacids

The starting dimethyl ester hydrochloride salt (1.0 equiv), the productof Example Preparation 2 was suspended in dry dichloromethane (0.1 Msolution) under nitrogen. The corresponding N-BOC-aminoacid (1.5 equiv),N-ethyl-N′,N′-dimethylaminopropylcarbodiimide (EDC, 1.5 equiv) and1-hydroxybenzotriazole (HOBt, 1.5 equiv) were added in one portion,followed by triethyl amine (1.0 equiv) via syringe and, finally,dimethylaminopyridine (DMAP, 0.1 equiv). The reaction mixture wasstirred overnight at room temperature, then hydrolyzed by addition of 1Nhydrochloric acid (20 ml/mmol) and diluted with methylene chloride (10ml/mmol). The aqueous layer was extracted with methylene chloride (5ml/mmol) and the combined organic layers washed twice with 1 Nhydrochloric acid (10 ml/mmol), and finally with water and brine (10ml/mmol each). After drying over sodium sulfate and evaporation undervacuum the crude residue was purified by silica gel chromatography usingthe appropriate eluent (typically mixtures hexanes/ethyl acetate).

Alternative procedure for the coupling reaction of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride with N-BOC-aminoacids

A solution of dicyclohexylcarbodiimide (DCC) (1.1 equiv) in methylenechloride (4.0 M solution) was added to a mixture of Preparation 2 (1.0equiv), triethylamine (1.0 equiv) and N-t-butoxycarbonyl-L-alanine (1.1equiv) in methylene chloride (1.0 M solution) over a period ofapproximately 1.5 h while stirring. The resulting mixture was stirredfor 1-12 h then filtered. The filter cake (dicyclohexylurea) was rinsedwith methylene chloride, and the filtrate was washed with 0.1 M NaHCO₃followed by 1.0 N hydrochloric acid. The organic phase was dried(Na₂SO₄), filtered and concentrated to afford the title compound as anoil.

EXAMPLE 1 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester

The starting dimethyl ester hydrochloride salt (1.0 equiv), the productof Preparation 2 was suspended in dry dichloromethane (0.1 M solution)under nitrogen. N-BOC-(L)-alanine (1.5 equiv),N-ethyl-N′,N′-dimethylaminopropylcarbodiimide (EDC, 1.5 equiv) and1-hydroxybenzotriazole (HOBt, 1.5 equiv) were added in one portion,followed by triethyl amine (1.0 equiv) via syringe and, finally,dimethylaminopyridine (DMAP, 0.1 equiv). The reaction mixture wasstirred overnight at room temperature, then hydrolyzed by addition of 1Nhydrochloric acid (20 ml/ mmol) and diluted with methylene chloride (10ml/mmol). The aqueous layer was extracted with methylene chloride (5ml/mmol) and the combined organic layers washed twice with 1 Nhydrochloric acid (10 ml/mmol), and finally with water and brine (10ml/mmol each). After drying over sodium sulfate and evaporation undervacuum the crude residue was purified by silica gel chromatography usingmixtures of hexanes/ethyl acetate.

50% Yield. Foamy white solid. mp 51-52° C. [α]_(D) ²⁵=−27.7 (c=0.52,CHCl₃). ¹H NMR (CDCl₃) δ: 7.28 (brs, 1H), 5.04 (brd, 1H, J=7.6 Hz), 4.16(m, 1H), 3.74 (s, 3H), 3.66 (s, 3H), 2.49 (dd, 1H, J=13.9, 8.3 Hz), 2.42(dd, 1H, J=6.3, 2.8 Hz), 2.18-1.89 (m, 3H), 1.70 (t, 1H, J=2.9 Hz), 1.45(s, 9H), 1.33 (d, 3H, J =7.0 Hz), 1.19 (m, 1H). ¹³C NMR (CDCl₃) δ:172.8, 172.6, 172.6, 155.7, 80.2, 66.3, 52.6, 51.8, 49.5, 34.4, 32.0,28.2, 28.1, 26.6, 21.1, 17.6.

Alternative Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester

A solution of dicyclohexylcarbodiimide (DCC) (1.1 equiv) in methlyenechloride (4.0 M solution) was added to a mixture of Example Preparation2 (1.0 equiv), triethylamine (1.0 equiv) andN-t-butoxycarbonyl-L-alanine (1.1 equiv) in methlyene chloride (1.0 Msolution) over a period of approximately 1.5 h while stirring. Theresulting mixture was stirred for 1-12 h then filtered. The filter cake(dicyclohexylurea) was rinsed with methlyene chloride, and the filtratewas washed with 0.1 M NaHCO₃ followed by 1.0 N hydrochloric acid. Theorganic phase was dried (Na₂SO₄), filtered and concentrated to affordthe title compound as an oil.

EXAMPLE 2 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

A solution of 2 M NaOH (5.45 L, 10.9 mol) was added to a solution of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester (4.52 mol, crude) in THF (2.8 L). The resultingmixture was stirred at ambient temperature for 3 h then extracted withCH₂Cl₂ (2×3 L). Ethyl acetate (5 L) and tetrahydrofuran (3 L) were thenadded to the aqueous phase. While stirring, concentrated HCl (970 mL)was added to the mixture until the pH=2. The organic phase was dried(MgSO₄) and filtered. The aqueous phase was then extracted with ethylacetate (5 L). The organic phase was dried (MgSO₄), filtered andcombined with the previous organic phase. The combined organics wereconcentrated to a soft solid. Ethyl acetate was then added, and themixture was concentrated to a soft solid. Ethyl acetate (3.5 L) wasagain added. The mixture was concentrated until a freely flowingsuspension was present. Heptane (1.8 L) was then added, and the slurrywas stirred at ambient temperature for 15 h. The solid was filtered,washed with heptane (3 L) then dried under vacuum to afford the titlecompound. Yield 1.36 kg (84%) as an approximate 85:15 mixture ofrotamers as a white solid. [α]_(D) 25-24.8 (C1.0, MeOH) ¹ H NMR(DMSO-d₆) δ 12.20 (s, 2H), 8.40 (s, 0.85H), 8.36 (s, 0.15H), 6.69 (d,J=8.2 Hz, 0.85H), 6.33 (br d, 0.15H), 3.99 (quintet, J=7.2 Hz, 0.85H),3.84 (br m, 0.15H), 2.18-2.13 (m, 2H), 1.91-1.84 (m, 1H), 1.82-1.75 (m,2H), 1.46 (br s, 0.85H), 1.43 (br s, 0.15H), 1.35 (s, 9H), 1.23-1.15 (m,1H), 1.13 (d, J=6.9 Hz, 3H). ¹³C NMR (CD₃OD) δ 176.4, 176.0 (2 C),157.5, 80.5, 67.3 (minor rotamer), 67.2 (major rotamer), 50.9, 35.6,32.8, 29.3, 28.7, 27.4, 22.1, 18.5. MS (EI) calcd for C₁₆H₂₈N₃O₇ (M+NH₄⁺) 374.20 found 374.24 m/z.

Alternative Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

A solution of(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (85 g, 418 mmol) and MeOH (850 mL) was cooled to 10° C.Thionyl chloride (199 g, 1.67 mol) was added at a rate such that thetemperature did not exceed 20° C. The solution was then heated to 50° C.and stirred for 6 h. Upon completion of the reaction, the solution wascooled to room temperature and concentrated to approximately 170 mLtotal volume under reduced pressure at 20-30° C. Water (850 mL) wasadded, and the pH of the solution was adjusted to approximately pH 2.0with 1.0 N NaOH (300 mL). The solution was concentrated under reducedpressure until the temperature reached approximately 40° C. Methylenechloride (850 mL) was then added, and the pH of the solution wasadjusted to pH 8 with 1.0 N NaOH (180 mL). The phases were separated,and the aqueous phase was extracted with CH₂Cl₂ (425 mL). The combinedorganic phases containing the corresponding dimethyl ester wereconcentrated to approximately 425 mL total volume and held for furtherprocessing.

In a separate reaction vessel a solution of N-t-butoxycarbonyl-L-alanine(83.2 g, 439 mmol) and 4-methylmorpholine (44.4 g, 439 mmol) in CH₂Cl₂(712 mL) was cooled to −5-−10° C. Isobutyl chloroformate (59.9 g, 439mmol) was then added at rate such that the temperature did not exceed−5° C. Upon completion of the addition, the solution was stirred for 15min. Simultaneously, CH₂Cl₂ (20 mL) was added to the dimethyl estersolution previously prepared, and this solution was cooled to −5° C. Thedimethyl ester solution (445 mL) was then added to the isobutyl mixedanhydride mixture. The cooling bath was removed, and the correspondingmixture was stirred for 30 min. A solution of 1.0 N HCl (445 mL) wasthen added. The phases were separated, and the organic phase was washedwith 1.0 N HCl (445 mL). The organic phase was concentrated toapproximately 180 mL total volume. THF (450 mL) was then added, and theresulting solution was concentrated to approximately 180 mL totalvolume. To this solution was added 1.0 N NaOH (1.67 L, 1.67 mol). Theresulting mixture was heated to 40° C., stirred for 1.5 h then cooled toroom temperature. Ethyl acetate (2.4 L) was added, and the pH of theaqueous phase was adjusted to pH 2.1 with concentrated HCl (150 mL). Thephases were separated, and the aqueous phase was extracted with ethylacetate (800 mL). The combined organic phases were dried with MgSO₄filtered and washed with EtOAc (2×320 mL). The resulting solution wasthen concentrated to approximately 400 mL total volume. Ethyl acetate(800 mL) was added, and the solution was concentrated to 400 mL). Thisethyl acetate addition/concentration was repeated again, then heptane(640 mL) was added. The resulting mixture was stirred for 2 h, filteredand washed with a 2:1 mixture of heptane-ethyl acetate (2×320 mL) toafford 115.5 g (78% yield) of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid as a white solid.

EXAMPLE 3 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

To a solution of ethyl acetate (500 mL) was added HCl (79.0 g, 2.16mol). The resulting HCl solution was then added to a slurry of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (100 g, 281 mmol) in ethyl acetate (500 mL) at a rate such that thetemperature did not exceed 25° C. The resulting mixture was stirred for3.5 hours then filtered affording 82.6 g of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride as an amorphous, white solid. This white solid wasthen added to acetone (290 mL) and water (57 mL). The resulting mixturewas heated to 48-52° C., and water (6.4 mL) was added until all of thesolid dissolved. Acetone (2.2 L) was added to the resulting solutionover a period of approximately 1 h. When the addition of acetone began,the heating mantle was removed. After the addition was complete, themixture was cooled to 0-−10° C. and stirred for 4 h. The mixture wasthen filtered and washed with cold acetone (75 mL) affording(1S,2S,5R,6S)-2-[(2′S)-(2′-amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride that was dried under vacuum at 40° C. to provide 76.6g (93% yield) of the title compound as a white, crystalline solid. 72%Yield. White crystalline solid. mp>250° C., dec. [α]_(D) ²⁵=−7.80 (c=1.0MeOH). ¹H NMR (Methanol-d₄) δ: 3.96 (q, 1H, J=7.0 Hz), 2.47 (dd, 1H,J=6.3, 2.7 Hz), 2.37 (dd, 1H, J=13.6, 8.2 Hz), 2.18-1.92 (m, 3H), 1.66(t, 1H, J=3.0 Hz), 1.53(d, 3H, J=7.0 Hz), 1.46-1.34 (m, 1H). ¹³C NMR(Methanol-d₄) δ: 175.2, 174.7, 170.2, 66.4, 49.0, 36.6, 32.0, 28.5,26.3, 21.2, 16.6. 80% Yield. White solid.

EXAMPLE 4 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methanesulfonate

A solution of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (1.07 g, 3.00 mmol), methanesulfonic acid (584 mL, 9.00 mmol) anddioxane (10 mL) was stirred for 48 h. The mixture was filtered and driedto afford (1S, 2S, 5R, 6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid methane sulfonate as a crude, white,amorphous solid (1.05 g). A sample of this solid (1.0 g) was dissolvedin MeOH (10 mL). The solution was concentrated to 3.3 g total weight andseed crystals were added. Ethyl acetate (10 mL) was then added to themixture over a period of 15 min. The mixture was stirred for 30 min,filtered and dried under vacuum to afford 830 mg of the title compoundas a white, crystalline solid. Yield 78% ¹H NMR (CD₃OD) δ 3.96 (q, J=7.1Hz, 1H), 2.71 (s, 3H), 2.45 (dd, J=6.4, 2.7 Hz, 1H), 2.38 (dd, J=13.9,8.4 Hz, 1H), 2.20-2.08 (m, 1H), 2.01-1.93(m, 2H), 1.67 (t, J=2.9 Hz,1H), 1.52 (d, J=7.0 Hz, 3H), 1.46-1.35 (m, 1H) ¹³C NMR (CD₃OD) δ 176.3,175.7, 171.2, 67.4, 50.0, 39.5, 35.7, 33.1, 29.5, 27.4, 22.2, 17.6.Anal. Calcd for C₁₂H₂₀N₂O₈S: C, 40.90; H, 5.72; N, 7.95. Found: C,40.81; H, 5.69; N, 7.83.

EXAMPLE 5 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

(1S, 2S, 5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride (1.0 g, 3.42 mmol) was dissolved in water (1 mL), and1.0 N NaOH (3.42 mL, 3.42 mmol) was added. The solution was maintainedin the refrigerator for 24 h. The solution remained clear. Acetone (2mL) was added, and the solution was stored in the refrigerator for 16 h.A white solid precipitated out of solution, and mixture could not bestirred. Acetone (4 mL) was added, and the mixture was stirred at rt,then filtered and dried to afford 630 mg of the title compound as awhite crystalline solid which contained 2-4% NaCl. Yield 72% ¹H NMR(CD₃OD) δ 3.93 (q, J=7.1 Hz, 1H), 2.48 (dd, J=6.6, 2.9 Hz, 1H), 2.32(dd, J=13.5, 8.4 Hz, 1H), 2.20-2.08 (m, 1H), 2.01-1.90 (m, 2H), 1.61 (t,J=2.9 Hz, 1H), 1.51 (d, J=7.0 Hz, 3H), 1.48-1.33(m, 1H) ¹³C NMR (CD₃OD)δ 176.9 (2 C), 171.1, 68.0, 50.1, 35.9, 33.2, 29.7, 27.3, 22.5, 17.6.

1. A method for treating anxiety in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of formula I

wherein R¹³, R¹⁴ and R¹⁷ is hydrogen; or a pharmaceutically acceptablesalt thereof.
 2. The method of claim 1 wherein the pharmaceuticallyacceptable salt of a compound of formula I is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride salt.
 3. The method of claim 1 wherein thepharmaceutically acceptable salt of a compound of formula I is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methane sulfonate salt.